2.1. Monitoring and Collection of Termites

For the monitoring and collection of C. formosanus and R. speratus termites, twelve “Substec^TM station mini” (SC Environmental Science, Osaka, Japan) stations containing wood were used for termite baiting (Figure 1) around three stumps (1, 2, and 3) at the test area in Isogi Park (33°39′35.7″ N, 135°20′34.4″ E, Wakayama Prefecture, Japan) in June 2016 (Figure 2). Four additional stations were installed around a fourth stump in July 2016. Three and four stations were additionally installed around stumps 1 and 4, respectively, in December 2016. Eight stations were installed around additional stumps, 5 and 6, in March 2017. For the monitoring and collection of R. speratus termites, thirty “Substec^TM station mini” stations containing wood were used for termite baiting at the test area on the Nara campus of Kindai University (34°40′18.5″ N, 135°44′03.4″ E, Nara Prefecture, Japan) in June 2013 (Figure 3). “Substec^TM wood mini” (SC Environmental Science, Osaka, Japan) wood inserts were used to monitor termite activity and were periodically inspected for four years, from 2016 to 2020, in Isogi Park and for six years, from 2013 to 2019, at Kindai University.

“Substec^TM station mini” with lid open and wood bait inside. Left: top view; Right: side view.

Location of monitoring stations (open circles) in Isogi Park. Twelve stations (1-1, 1-2, 1-3, 1-4, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-3, and 3-4) were installed around stumps 1, 2, and 3 in June 2016. Four stations (4-1, 4-2, 4-3, and 4-4) were installed around stump 4 in July 2016. Three stations (1-5, 1-6, and 1-7) and four stations (4-5, 4-6, 4-7, and 4-8) were additionally installed around stumps 1 and 4, respectively, in December 2016. Eight stations (5-1, 5-2, 5-3, 5-4, 6-1, 6-2, 6-3, and 6-4) were installed around stumps 5 and 6 in March 2017. All stations were installed in-ground. Station 4-1, around which discontinuous soil treatment was carried out, is highlighted in blue.

Location of monitoring stations (open circles) at Kindai University. All stations were installed in-ground in June 2013. Stations 1–7 were installed in flat ground. Stations 8–17 and 18–22 were set up at the foot of a hill and along the path, respectively. Stations 23–26 and 27–30 were installed around the stumps. Station 5, around which discontinuous soil treatment was carried out, is highlighted in blue.

For microsatellite genotyping, between three and 19 workers and/or soldiers (n values, Table 1, Table 2 and Table 3) were collected from stations or stumps during inspection periods. For residue analysis of fipronil and its degradation products (Figure 4), between one and 14 C. formosanus foragers (n values, Table 4) were collected from Isogi Park stations one week after the soil treatment described below. Between five and 163 R. speratus foragers (n values, Table 5 and Table 6) were collected from the Kindai site four days and three weeks after discontinuous soil treatment with fipronil (see below). Specimens for both DNA genotyping and residue analysis were stored at −20 °C until extraction.

The degradation of fipronil to fipronil sulfide, fipronil sulfone, and fipronil desulfinyl occurs via reductive, oxidative, and photolytic mechanisms, respectively.

Statistics of microsatellite loci and F-statistics for Coptotermes formosanus cohorts collected in Isogi Park from November 2016 to May 2020.

¹ Cohort: notated as a combination of station number plus collection date; bold: cohorts found by the time of discontinuous soil treatment; n: sample size; N_A: number of alleles; F_IS: coefficient of inbreeding for individuals within their subpopulations; F_IT: coefficient of inbreeding for individuals relative to the total population; F_ST: coefficient of inbreeding of subpopulations relative to the total population; He: expected heterozygosity; Ho: observed heterozygosity.

Statistics of microsatellite loci and F-statistics for Reticulitermes speratus cohorts collected in Isogi Park from October 2018 to September 2020.

¹ Cohort: notated as a combination of station number plus collection date; n: sample size; N_A: number of alleles; F_IS: coefficient of inbreeding for individuals within their subpopulations; F_IT: coefficient of inbreeding for individuals relative to the total population; F_ST: coefficient of inbreeding of subpopulations relative to the total population; He: expected heterozygosity; Ho: observed heterozygosity.

Statistics of microsatellite loci and F-statistics for Reticulitermes speratus cohorts collected at Kindai University from September 2013 to November 2019.

¹ Cohort: notated as a combination of station number plus collection date; bold: cohorts found by the time of discontinuous soil treatment; n: sample size; N_A: number of alleles; F_IS: coefficient of inbreeding for individuals within their subpopulations; F_IT: coefficient of inbreeding for individuals relative to the total population; F_ST: coefficient of inbreeding of subpopulations relative to the total population; He: expected heterozygosity; Ho: observed heterozygosity.

Fipronil and its sulfide, sulfone, and desulfinyl derivatives detected in worker and soldier caste termites of Coptotermes formosanus collected in Isogi Park one week after the soil treatment.

¹ Detection limit for each sample. ² Values below the detection limits are shown as 0 even if trace amounts were quantified. n: sample size, with weight of termites in parentheses.

Fipronil and its sulfide, sulfone, and desulfinyl derivatives detected from worker caste termites of Reticulitermes speratus collected at Kindai University four days after the soil treatment.

¹ Detection limit for each sample. ² Values below the detection limits are shown as 0 even if trace amounts were quantified. n: sample size, with weight of termites in parentheses.

Fipronil and its sulfide, sulfone, and desulfinyl derivatives detected from worker caste termites of Reticulitermes speratus collected at Kindai University three weeks after the soil treatment.

¹ Detection limit for each sample. ² Values below the detection limits are shown as 0 even if trace amounts were quantified. n: sample size, with weight of termites in parentheses.